Endocannabinoids measurement in human saliva as potential biomarker of obesity

Abstract

Background: The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/principal findings: Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB(1)) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands. The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB(1) receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/significance: Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.

Figure 1. Fasting salivary endocannabinoids and N-acylethanolamines are elevated in obese subjects.

Fasting salivary and plasma AEA (A), 2-AG (B), PEA (C) and OEA (D) levels in normal weight and obese subjects. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Relationship of salivary AEA and OEA levels with anthropometric and metabolic parameters.

Fasting salivary AEA and OEA correlation with body mass index (BMI) (A, D), waist circumference (B, E) and fasting insulin levels (C, F).

Figure 3. The ECS is present in human salivary glands.

(A) RT-PCR for CB₁, FAAH, MAGL, DAGL alpha, DAGL beta and housekeeping genes GAPDH and HRPT performed on submandibular and accessory salivary glands. (B) RT-PCR for NAPE-PLD and NAAA performed on submandibular and accessory salivary glands. A 100-bp ladder is shown. (C) Endocannabinoids and related N-acylethanolamines content in submandibular salivary glands. CB₁: Cannabinoid receptor type 1, DAGL alpha: Diacylglycerol lipase alpha, DAGL beta: Diacylglycerol lipase beta, FAAH: Fatty acid amide hydrolase, GAPDH: glyceraldehyde-phosphate dehydrogenase, HPRT: hypoxanthine phosphoribosyl transferase, MAGL: Monoacylglycerol lipase, NAAA: N-acylethanolamine-hydrolyzing acid amidase, NAPE-PLD: N-acyl phosphatidylethanolamine phospholipase D.

Figure 4. Changes in salivary endocannabinoids and N-acylethanolamines levels in response to a meal.

Salivary AEA (A), 2-AG (B), OEA (C) and PEA (D) levels 1 h before (1 h PRE), immediately before (PRE) and 1 h after the termination of the meal (1 h POST). *p<0.05 normal weight vs. obese at a specific time point; ^# p<0.05, ^## p<0.01 normal weight vs. obese.

Figure 5. Changes in salivary endocannabinoids and N-acylethanolamines levels in response to body weight loss.

Salivary AEA (A), 2-AG (B), PEA (C) and OEA (D) before and after body weight loss obtained through a 12 weeks life-style intervention program. *p<0.05 before vs. after the lifestyle intervention program.